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Re: Cloning prehistoric animals...
Ah, I see I should apologize, you do have a grasp of the technology and the
workings of DNA. I am often in the position of explaining simple things like the
structure of DNA to students and postdocs and sometimes forget that not everyone
is unaware.
Having said that, I do have a few comments and questions.
> My apologies. I thought that PCR usually used macroscopic samples of DNA and
> magnified the signal by duplicating the molecules, not reading molecules. I
> did not know that technology was available to pick a single molecule and
> read it directly, without duplication. I know that PCR will work with a
> single piece of a DNA strand, but picking the right stuff will be hard
> unless you can read a molecule without going through that process.
Little nitpicky point, macroscopic samples of DNA would overwhelm a PCR
reaction. We often use nanogram amounts. In many instances, the less DNA (to a
certain point), the better the PCR actually works.
I don't understand the necessity of reading a single strand without duplicating
it. No, we don't do that, true. We duplicate it a lot and that seems to work
very well. I can understand the concern that with replication there is the
chance that mistakes are going to be made, but these can be minimized so that
the chances of a mistake affecting an active part of a gene is very small.
If you are concerned about contamination with DNA from other sources, again,
yes, that is a problem. But you would have that problem no matter what technique
you used. It can be also be reduced by proper handling of the sample and
solutions and can be checked by comparisons with known sequences.
>
>
> Your note says "if we can find complete strands" but my understanding is
> that even the human genome project, with tons of great, fresh material
> (moles of complete strands) and huge resources (>$10^9) has crowed "success"
> when they analyzed only about 90% or less of a single human chromosome
> (Nature, vol. 402, p 489, 12/2/99). With much less material and the amount
> of degradation expected in the ancient samples, it doesn't look good for
> mammoths.
True, but the paper was not so much a milestone in what has been achieved, but a
proof of technique. The percentages published in the paper were also
conservative. The authors actually think think they have correctly sequenced
much more than 90% and virtually all the genes of that chromosome. We can expect
that the remaining chromosomes will be finished much more quickly. It would not
be unreasonable to guess that the entire genome will be sequenced within three
years. Dr. Venter of The Institute for Genomic Research has stated that they
should be finished with 90% within two. The technology to sequence vast amounts
of DNA has grown so staggeringly in the past couple of years it has been hard to
keep up with. The first complete genome of a microorganism was only finished in
1995. We now have several dozen if not hundreds done as well as some eukaryotes,
such as the fly Drosophila melanogaster and the nematode Caenorhabditis elegans.
The technology has truly undergone major revolutions in only a couple of years.
But, you are right, all these projects had plenty of good quality starting
material. Still, if ever there was a science that has shown great promise in its
earliest infancy, which is what this field is in, I think molecular biology
would qualify as one of the tops of the list. With more work, the easier it will
be to develop sequences from much less starting material and less good quality.
>
>
> With regard to hydrogen bonding, I was trying to suggest that the statistics
> of degraded spots on the molecules that had survived would be influenced by
> the local strength of the bonds between strands, i.e. that areas of the
> molecule that unzipped would degrade faster. If this occurs it will produce
> a spatial correlation in the degradation funciton, i.e.the places that
> degraded most through time would be pretty much the same from molecule to
> molecule, so that even if one has a large sample of molecules, one does not
> have full information.
Ah, the longer explanation makes sense to me now. Yes, I agree. That is quite
true. A problem to be sure. I would hypothesize that the most likely places for
that to happen, however, will be identical between the mammoth and the elephant
and could most likely then be replaced with elephant genes. This assumes a
limited degradation, though, which could be completely mistaken. Nevertheless, I
do think that with the condition of the mammoth corpse as I understand it, it
should be possible to find reasonably intact DNA strands. There should not be
that many genes separating mammoths and elephants, so it should be feasible
theoretically.
This is still a huge undertaking and not one I expect will happen anytime soon,
but it seems to me technologically feasible.
>
>
> I understand that the bonds along the strand are covalent. I also understand
> that the information on the DNA is redundant and that only one side need be
> available, but I don't believe that the complete information will be
> available from sources of ancient DNA because of the correlation of degraded
> spots.
True, but the mammoth is not that old and is relatively good condition. It might
be possible from it. Not holding my breath for dino DNA unfortunately.
> Unless single strands with excellent preservation in critical spots
> can be selected (by a technology I believe may lie in the future), the
> dominant input signal in a PCR sample taken from a macroscopic sample will
> have correlated bad spots and will overwhelm the small fraction of molecules
> with preserved information in the critical areas.
I can envision a way to do just that but I would want to test it out to figure
out how to best deal with it before I even thought about playing around with
precious irreplaceable DNA. But basically, it is possible to run a PCR to
determine the most common strands and remove them leaving only the rarer
sequences, then repeat the process until you have a subset of very rare
sequences. You can then use them to rebuild the total strand. It can be done in
principle, but I have not tested it in actual practice. Want to give me a few
hundred thousand and a year or two?:)
Sorry if I came off too brusque or condescending.
I am also feeling a premonition that Mickey or Betty might be telling us soon to
wrap up this conversation or take it offlist as it is rather straying away from
the topic of dinosaurs, though.
Joe Daniel